1. Field of the Invention
The present invention relates generally to the field of detection of viral infection. Specifically, the invention relates to the early detection of human immunodeficiency virus (HIV) infection. Even more particularly, the invention relates to the early detection of HIV infection through the detection of antibodies directed against a preparation of non-denatured HIV envelope antigens. The invention also relates to the characterization of an HIV antigen preparation immunoreactive with these early anti-HIV antibodies, and methods of using them in anti-HIV antibody detection.
2. Description of the Relevant Art
A retrovirus is currently believed to be etiologically associated with acquired immunodeficiency syndrome (AIDS). (3, 5). The retroviruses involved are members of the Lentivirus family, variously termed LAV, HTLV-III, and ARV, and collectively termed human immunodeficiency virus (HIV) (3). These virus are commonly transmitted via blood or blood products (1-4), as well as through sexual contact or use of hypodermic needles shared with persons infected with the virus (4). Spread of the HIV virus has now reached epidemic proportions, thus prompting scientists and clinicians around the world to concentrate on developing methods to prevent its continued spread.
A variety of detection or "screening" methods have been developed to control the spread of the virus. For example, some clinicians sought to determine the presence of live virus in patient samples suspected of having been infected (5) However, it was soon found that the virus was not easily isolatable from patient samples, with as high as 40% of infected persons going undetected (5). The low level of detectability by this method is attributable to the typically low circulating concentrations of the virus or virus-infected cells, especially in early infected persons and those in whom anti-viral antibodies were present. In such patients, low levels of circulating virus would be expected owing to the neutralizing activity of the antibody (46). High percentages of false negative HIV exposure diagnoses inevitably resulted. Additionally, the isolation of live virus was very time consuming and expensive, and not readily adaptable to routine laboratory procedure, being limited by the hazardous nature of live virulent cultures.
An alternative procedure which developed to monitor HIV infections was through the detection of various molecular weight denatured HIV virus antigens. The first available assays for blood antigen screening were based on the antigen-capture system. The sensitive detection and quantitation of antigen was pioneered by Berson et al. (45) with the development of the radioimmunoassay (RIA). The RIA is an inhibition assay in which unlabeled antigen (test antigen) competes with a labeled antigen for limited antibody. Some of the more important modifications made to the original RIA were the use of labeled antibodies rather than labeled antigen (39) and the use of solid phase absorbants. The RIA is a useful assay but depends on a principle (competition of two antigens for antibody) that cannot be reversed for the accurate measurement of antibodies.
The presence of HIV DNA (i.e., HIV DNAn) was also tested by enzymatic amplification of HIV-1 DNA using the polymerase chain reaction method. Loche et al. were able to detect HIV DNA through the use of a direct diagnostic test based on hybridization to amplified viral DNA in serum samples 6-14 months before seroconversion as documented by conventional methods (16). Over 23% of a group of persons testing negative (31 of 133) for anti-HIV antibodies by conventional enzyme-linked immunosorbent assay (ELISA) and Western Blot were identified as positive for the presence of HIV antigen as well as HIV DNA by the above methods (17).
Recently, some laboratories have been able to develop antigen detection assays with enhanced sensitivity (19). These assays detected HIV antigen up to three weeks prior to that of conventionally practised antigen detection ELISA's of the day, and with a reduced assay time (i.e., four hours vs. "overnight" incubation) (19). However, such systems measured only denatured HIV antigen of various molecular weights, and were still plagued with high percentages of false-negative diagnoses. The disadvantage of detecting denatured viral antigen is that by the time these antigens are detectable in a patient, the infection has already progressed to its more advanced stages. Cumulative studies by those of skill in the art indicate that a more antibody-sensitive "antigen" or an alternative test method is needed to be developed and used if consistent and accurate early HIV exposure is to be achieved.
While detection of antigen appeared to result in a more reliable diagnosis than live-virus isolation, the need still existed for a more sensitive assay capable of detecting anti-HIV antibodies during early stages of HIV exposure. Quantitation of antibodies is a major objective and problem in immunology since absolute units of antibodies are often difficult to estimate because of the influence of antibody affinity on measurements (39). The ELISA was proposed as the antibody detection test of choice as it provided for the convenient testing of large numbers of serum samples at a time. However, the high frequency of false positive results using denatured HIV-antigen as "target" antigen required further refinement of the assay, as well as a more sensitive "target" antigen to enhance sensitivity of antibody detection (5).
Some investigators have proposed the use of alternative HIV-antigen preparations in standard anti-HIV antibody screening tests (8). The presence of antibodies to gp160 and gp120 (RIP) have been described as the most consistent indicators of HIV-1 exposure in hemophiliacs and in homosexuals (10-11). Chou et al. sought to detect anti-HIV antibody using an infected cell lysate (RIP) or viral lysate (WB) preparation of gp120 HIV with a non-reducing buffer as the "target" antigen. Sensitivity of antibody to gp120 HIV "target" antigen prepared with nonreducing buffer was enhanced by an additional 25% (9). However many persons continued to be reported as "seronegative" for anti-HIV antibodies despite detectable levels of circulating virus. A significant proportion of infected persons with detectable circulating live virus continued to be described as immunologically "seronegative".
Monitoring HIV exposure through antibody detection is plagued by the observation that only very low titers of circulating anti-HIV antibodies are observed in patients in whom detectable circulating levels of live virus are found (12-13). While these and other tests, such as radioimmuno-precipitation and SDS-PAGE (RIP), have led to a decrease in the percentage of reported false negative and false positive diagnoses, there still existed difficulties in determining seropositivity of vague antibody profile patterns (7-8).
A variety of theories were set forth to explain the phenomena of HIV infection without the presence of detectable anti-HIV antibody. One explanation was that in sexually transmitted HIV infection, a long period of latency occurred during which the virus was present, but remained dormant and did not invoke an initial production of detectable anti-HIV antibodies (i.e., pre-seroconversion) (15). Imagawa et al. describe this "silent" infection (i.e. no detectable anti-HIV antibodies by conventional methods) as extending up to 36 months after the positive culture of live HIV virus (17). During this "latency period", persons were described as seronegative for HIV antibody (17).
Haseltine postulates that the failure to detect anti-HIV antibodies in persons with HIV virus indicates the establishment of HIV-1 infection without triggering the immune system, and that loss of antibodies may occur after transient production of the virus and antiviral antibodies, a so-called "silent infection" period (40). This "silent infection" period has been reported to linger years before antibodies are detectable by current methods (18). The state of supposed "silent infection" in which virus is detectable without anti-viral antibodies is foreshadowed by earlier reports on high risk, homosexual men, in whom low-titer antibodies were detected, also having virus and/or viral nucleic acids (16).
An alternative method for detecting antibodies is live-cell immunofluorescence. The use of live-cell immunofluoresence has been applied in the analysis of surface antigens of Friend murine leukemia virus-infected (F-MuLV) cells and gibbon ape lymphoma virus infected cells (35). This technique offers the advantage of direct visualization of bound antibody on the surfaces of viable cells. Such assays were adapted for containment of these potentially hazardous virus infected cells (Id.) Applicants later developed a live-cell membrane immunofluorescence assay for the analysis of HIV infected cell surface antigenic determinants (36).
Despite intensive studies into the pathology of HIV, the detection of anti-HIV antibodies in samples obtained months or years prior to classically detectable "seroconversion" in patients from whom live virus is isolated still eludes investigators. It has been assumed that failure of conventional methods (WB, ELISA) to detect viral antigen or anti-HIV antibodies indicates that no antibodies are being produced against the virus. However, false-negative tests for anti-HIV antibody continues to result in failure to detect patients with early transmittable HIV infections. Thus, non-detected HIV-contaminated blood units obtained from persons during the early stages of HIV infection continue to threaten blood recipients of all ages, sexes and sexual preferences.
The development of a new generation serological test able to detect anti-HIV antibodies during the early phases of HIV infection would provide clinical monitoring laboratories and hospitals throughout the world an effective method for halting the spread of AIDS and AIDS-related conditions heretofore frustrated by the complex immunology and biochemical nature of the human immunodeficiency virus.